JPH095289A - Two-dimensional sensor for measuring activity of nerve cell and measuring device therewith - Google Patents

Abstract

PURPOSE: To provide a two-dimensional sensor for measuring activity of a nerve cell which is excellent in general usefulness and a measuring device therewith by making the position or dimension of a measuring electrode to be variable. CONSTITUTION: A two-dimensional sensor 1 is provided with a thin film 1a for working electrode which is vapor-deposited to the rear surface of Si side of a sensor board comprised of three layers, Si, SiO2 and Si3 N4 , and an enclosure 1b to house a cell as a sample, a culture solution and a reference electrode on the surface of Si3 N4 side thereof, and it is placed within an incubator 3, then a bias voltage is applied to between the reference electrode and the working electrode. A high-frequency-modulated laser beam is emitted from the rear of the sensor 1, and a signal whose potential is changed by cell activity at the irradiated portion is picked up as an optical AC current, then it is inputted into a computer 10 through an amplifier 9.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a two-dimensional sensor for measuring cell activity by detecting potential changes associated with activity of nerve cells and the like, and a measuring device using the same.

[0002]

2. Description of the Related Art In recent years, medical examination of nerve cells and possibility of application as an electric element have been actively conducted. An action potential is generated when a nerve cell is activated.
This action potential is caused by a change in the ion concentration inside and outside the cell membrane accompanying a change in the ion permeability of the nerve cell, and a change in the cell membrane potential accordingly. Therefore, when observing the activity state of sample cells or tissues,
By measuring the above-mentioned two-dimensional distribution of cell membrane potential, it is possible to identify the active site and observe the degree of activity.

In the measurement of such a cell potential, a so-called two-dimensional sensor that enables measurement without inserting a so-called glass or an electrode for giving a stimulus into a cell, and at the same time, can measure a plurality of points on a plane. There is an integrated composite electrode previously filed by the applicant (Japanese Patent Application Laid-Open No. 6-7
8889, JP-A-6-296595). This integrated composite electrode is formed by forming a plurality of microelectrodes arranged in a matrix using a conductive substance on a glass substrate and drawing patterns thereof, and enables cell culture on this. As a result, compared to the case where a glass electrode or the like is used, it is possible to simultaneously measure the potential change at a plurality of places at narrow intervals, and further, it is possible to observe it for a long time while culturing cells / tissues.

[0004]

However, the conventional integrated composite electrode as described above lacks versatility because the position and size of the measuring electrode are fixed, and is also used for measuring the activity of various cells and tissues. It was difficult to do. Actually, the intervals and sizes of the measurement electrodes were optimized by experiments according to the cells to be measured, and an almost dedicated integrated composite electrode was created.

Therefore, the present invention improves the conventional integrated composite electrode described above and makes the position of the measuring electrode variable so as to provide a versatile two-dimensional sensor for measuring cell activity and a measuring device using the same. The purpose is to provide.

[0006]

The features of the two-dimensional sensor for measuring cell activity according to the present invention are Si, SiO ₂ , and Si _3.
Thin film for the working electrode on the back surface of the Si side of the sensor substrate made of three layers of N ₄ is deposited, the cell as a sample on the surface of the Si ₃ N ₄ side, broth, and the enclosure to put a reference electrode is provided, that In the plane distribution of the potential change generated by the activity of cells placed in the enclosure, a signal mainly corresponding to the potential change in the portion irradiated with light is extracted from the working electrode.

A feature of the cell activity measuring apparatus according to the present invention using the above two-dimensional sensor is that the back surface of the two-dimensional sensor is irradiated with a laser beam and a working electrode on the back surface of the two-dimensional sensor. It is provided with a DC power source for applying a DC bias voltage between the electrode and the reference electrode placed in the enclosure on the surface side, and a processing means for processing a signal obtained between the both electrodes. A preferred specific configuration will be described later.

[0008]

The two-dimensional sensor for measuring cell activity according to the present invention is
LAPS (Ligh
t-Addressable Ptentiometric sensor, US Pat. No. 4,75
8786, No. 4,963,815, etc.) is applied to the measurement of cell potential. As shown in FIG. 5, the LAPS is formed by depositing an oxide film 102 on the semiconductor silicon substrate 101.
And a nitride film 103 are formed, and are used as, for example, a sensor for measuring the pH of a solution (electrolytic solution) 104 brought into contact therewith. The measurement principle is briefly touched upon.

Electrolyte, Insulato
When a bias voltage is applied to the EIS structure composed of r) and a semiconductor by a potentiostat 105 and light modulated at a certain frequency is irradiated from the back surface of the EIS structure, an optical alternating current as shown in FIG. 6 flows. At this time, I showing the relationship between the bias voltage V and the optical alternating current I
The −V curve shifts in the horizontal axis (bias voltage) direction as shown in the figure depending on the pH of the solution. Therefore, the pH of the electrolytic solution can be measured by detecting the photocurrent I while the predetermined bias voltage is being applied. I
The reason why the −V curve shifts depending on the pH of the solution is considered as follows.

When a voltage is applied to the EIS structure, a bending of the energy band occurs at the interface between the semiconductor and the insulating film, and this bending also depends on the pH of the solution in contact with the insulating film. In other words, silanol groups (Si-O
H) and an amino group (Si-NH ₂ ) are formed, but these functional groups selectively bond with the proton (H ⁺ ) and the equilibrium state between the proton present in the solution and the bound proton Has been maintained. Therefore, if the pH of the solution changes, the charge on the insulating film changes, which changes the bending of the energy band. As a result, the width of the depletion layer at the interface between the semiconductor and the insulating film, and hence the depletion layer capacitance, changes, and the flowing photocurrent also changes. This LAPS also utilizes the photoconduction phenomenon of a semiconductor whose electric conductivity is increased by irradiation with light.

The two-dimensional sensor for measuring cell activity according to the present invention, like the LAPS, has Si, SiO ₂ and Si _{3 as well.}
A sensor substrate composed of three layers of N ₄ is provided, and a thin film for a working electrode is vapor-deposited on the back surface on the Si side. However, the surface of the Si ₃ N ₄ side is provided with an enclosure for containing the sample cells, the culture solution, and the reference electrode, and the planar distribution of the potential change caused by the activity of the cells placed in the enclosure is directly measured. . That is, unlike the pH sensor using LAPS, the action of cells contacting the surface of the insulating film does not generate a potential on the surface of the insulating film due to the bond between the silanol groups and amino groups formed on the surface of the insulating film and the protons. By this, a potential is directly generated on the surface of the insulating film, which changes the width of the depletion layer at the interface between the semiconductor and the insulating film, and thus the capacitance. Then, since the electric conductivity of the portion irradiated with light becomes high, a signal mainly corresponding to the potential change of the portion can be taken out from the working electrode.

Therefore, as a structure of a cell activity measuring apparatus using such a two-dimensional sensor, a light source for irradiating the back surface of the two-dimensional sensor with a light beam, a working electrode on the back surface side of the two-dimensional sensor, and an enclosure on the front surface side. A power supply for applying a DC bias voltage between the reference electrode placed inside and a processing means for processing the signal obtained between both electrodes are required. The use of a laser light source as a light source is advantageous in that the irradiation spot can be accurately positioned and the spot diameter can be narrowed. Further, by providing the culture maintaining means for maintaining the culture environment of the cells placed in the enclosure of the sensor surface, the measurement for a long time becomes possible.

As a specific configuration for signal processing, a light source driving means for driving a laser light source at a high frequency to irradiate a high frequency modulated light is provided, and the processing means is provided for generating an optical alternating current flowing between a working electrode and a reference electrode. It is preferable to detect a change in amplitude. That is, as described above, a change in the width (capacitance) of the depletion layer at the interface between the semiconductor and the insulating film due to a change in potential caused by the activity of cells in contact with the surface of the insulating film is detected as a change in the amplitude of the photocurrent. .

Further, it is preferable that a means for scanning the laser beam emitted from the laser light source within a predetermined range on the back surface of the two-dimensional sensor (at high speed) is provided, whereby a plurality of locations within the predetermined range can be provided. Cell activity can be measured almost simultaneously. A laser array in which a plurality of laser elements that irradiate laser light substantially perpendicularly to the back surface of the two-dimensional sensor instead of scanning one laser beam are arranged in a two-dimensional matrix is provided. If is driven in a time-division manner, higher speed scanning becomes possible. Alternatively, instead of scanning the laser beam, X that controls the horizontal position of the two-dimensional sensor
A Y stage may be provided, and the position where the two-dimensional sensor irradiates the laser light may be changed by controlling the Y stage.

[0015]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 shows the configuration of an embodiment of the cell activity measuring apparatus according to the present invention. A cell 2 and a culture solution set on the two-dimensional sensor 1 for measuring cell activity are stored in the incubator unit 3.

The two-dimensional sensor 1 includes Si, SiO ₂ , and S as shown in the sectional view (a) and the plan view (b) of FIG.
A gold antimony thin film 1a for a working electrode is vapor-deposited on the Si-side back surface of a sensor substrate composed of three layers of i ₃ N ₄ , and an enclosure 1 for containing cells, a culture solution, and a reference electrode on the Si ₃ N-side surface.
b. The cross-sectional view of FIG. 2A is exaggerated in the thickness direction, and in actuality, the total thickness is 2
00 μm, of which SiO ₂ is 50 nm or less, Si ₃ N
₄ films are 100 nm or less. As the silicon (Si) substrate, an N-type of 10 ohm cm, a thickness of 200 μm, and a mirror-polished back surface was used. The gold antimony thin film 1a deposited on the back surface was alloyed at 500 ° C. to form an ohmic contact.

A silicon nitride film (Si
_{Since 3} N ₄ ) is non-invasive to living bodies such as cells, it is suitable for culturing cells and the like. The enclosure 1b containing cells etc. is
A polycarbonate cylinder having an inner diameter of 26 mm is adhered to the surface of a Si ₃ N ₄ film. In addition, machine screws for attaching an aluminum frame are fixed to four peripheral portions of the disc-shaped two-dimensional sensor 1.

Returning to FIG. 1, the incubator section 3 has a double structure, and it is sensible to prevent external contamination by various bacteria. Sample chamber 3 of incubator section 3
a is that the temperature control unit 4 controls the heater / fan unit 5 based on the output of the temperature sensor,
Maintained at 37 ± 0.5 ° C. In addition, a mixed gas adjusted to a ratio of 95% air and 5% carbon dioxide is introduced into the sample chamber. A flow meter 6 and a solenoid valve 7 are provided in the mixed gas supply path, and a timer 7b for turning on / off a drive circuit 7a for the solenoid valve 7 is also provided. To apply a bias voltage between the reference electrode (RE) placed in the enclosure of the two-dimensional sensor 1 in which the culture maintaining means is constituted by the incubator portion, the temperature control unit 4, etc., and the working electrode on the back surface. The potentiostat 8
Is provided, and the current signal flowing between both electrodes is input to the computer 10, which is a processing unit, via the amplifier 9. The computer 10 is equipped with a 16-bit A / D converter.

In the enclosure of the two-dimensional sensor 1, there is provided a counter electrode (CE) connected to the same potentiostat 8 as the reference electrode (RE) immersed in the culture solution.
By contacting the counter electrode (CE) with the sample and applying a pulse voltage between the counter electrode (CE) and the reference electrode (RE), the sample can be stimulated and the resulting evoked potential can be measured. This stimulation voltage (pulse voltage) is supplied from the computer 10 according to a command from the potentiostat 8
Occurs. Of course, it is also possible to measure the self-power generation position without giving a stimulus.

FIG. 1 also shows a laser light source 11 for irradiating the back surface of the two-dimensional sensor 1 with a laser beam and a driving device 12 for the laser light source 11. The laser beam emitted from the laser light source 11 is a mirror or lens (using an objective lens of an inverted microscope)
The beam is condensed to a beam diameter of about several μm by the optical system according to (3) and is irradiated on the back surface of the two-dimensional sensor 1. Also, the light source driving device 1
Reference numeral 2 includes a modulator that modulates the laser beam at a high frequency of several kHz to 10 kHz.

As a means for changing the position of the back surface of the two-dimensional sensor 1 irradiated with the laser beam, an XY stage 13 for horizontally displacing the two-dimensional sensor 1 together with the incubator unit 3 is provided. The XY stage 13 is capable of displacing the laser beam irradiation position of the two-dimensional sensor 1 in the XY direction in units of 1 μm by driving a step motor according to a command from the computer 10.

In this embodiment, the position of the laser beam is fixed and the two-dimensional sensor 1 is displaced in the XY directions as described above.
It is better to fix the position of the dimension sensor 1 and scan the laser beam. This can be achieved by using an XY galvanometer mirror in the optical system. Alternatively, a method of using a laser array in which a plurality of laser elements that irradiate a laser beam substantially perpendicularly to the back surface of the two-dimensional sensor are arranged in a two-dimensional matrix and driving the plurality of laser elements in a time division manner is also considered. To be

As described above, hole-electron pairs are generated in the portion of the two-dimensional sensor 1 irradiated with laser light, and photocurrent is generated by the bias voltage applied between the reference electrode and the working electrode. Is about to flow. Since the insulating film (SiO ₂ and Si ₃ N ₄ ) is formed on the surface of the two-dimensional sensor 1, a direct current does not flow, but an alternating current flows due to the high frequency modulation of the laser beam as described above. obtain. The capacitance of the insulating film (hereinafter simply referred to as capacitance) is C
i, the capacitance of the depletion layer at the interface between the semiconductor and the insulating film is Cd, and the photo-AC current generated in the depletion layer by photoexcitation by the high-frequency modulated laser beam is i _P. Photoacoustic current i flowing between the working electrode and
Is given by the following equation (Equation 1) from the equivalent circuit of FIG.

[0025]

Equation 1 the potential to _{i = Ci · i P / (} Ci + Cd) insulating film surface by activities of cells in contact with the insulating film surface is generated, whereby bending of the energy band is generated in the vicinity of the interface between the semiconductor and the insulating film, As a result, the width of the depletion layer at the interface between the semiconductor and the insulating film, that is, the depletion layer capacitance Cd changes. Then, the photo-acoustic current i detected outside also changes according to the above formula (Equation 1). In the case of the sensor using the N-type semiconductor as in the present embodiment, it is known that the depletion layer capacitance Cd increases and the photo-AC current i decreases when the potential generated on the surface of the insulating film is positive.
On the contrary, if the potential generated on the surface of the insulating film is negative, the depletion layer capacitance Cd becomes small and the photo-AC current i becomes large.

An example of an experiment in which the neural activity of a rat brain slice is observed using the above measuring device will be described below. The brains of 2-day-old postnatal SD rats were excised, and the visual cortex was cut into about 0.5 mm thick sections. This brain section was cultured in the enclosure of the two-dimensional sensor. The silicon nitride film on the sensor surface was treated with polylysine to improve the adhesion of the slices, and DF + f was used as the medium. Here, DF is a 1: 1 mixture of DMEM and Nutrient Mixture (F12 medium), and f is insulin 5 μg / m 2.
l, transferrin 100 μg / ml, progesterone 20nM, hydrocor
tisone 20 nM, putresine 100 μM, selenium 20 nM, and 5% fetal calf serum were added.

Such a sample (brain slice of a rat aged 1 to 2 days) causes spontaneous ignition within 1 week to 10 days after the start of culture. FIG. 4 shows the electrical activity of the sample detected by the above measuring device. FIG. 4A shows an optical AC current waveform digitally sampled at 48 kHz. FIG. 4B shows a waveform obtained by averaging the amplitude of this optical AC current every 10 msec. As can be seen from these figures, about 144.8 seconds to 145.
The amplitude of the optical alternating current is reduced by about 5% in 2 seconds.

From this, it is considered that a positive potential is generated on the surface of the insulating film due to the activity of nerve cells at the portion irradiated with the laser beam. Even when the irradiation location of the laser beam was moved to another location, a similar phenomenon of the amplitude of the photocurrent was observed at the location where spontaneous firing of the nerve cells was considered to occur.

[0029]

As described above, according to the present invention,
LAPS consisting of three layers of Si, SiO ₂ and Si ₃ N ₄
A two-dimensional sensor with a structure is used, and the cells of the sample can be cultured on the two-dimensional sensor substrate so that the potential change due to the cell activity at the portion irradiated with the laser beam from the back surface of the two-dimensional sensor substrate can be detected. By changing the beam irradiation position and the spot diameter, the position and size of the measurement electrode can be adjusted almost arbitrarily. That is, it was possible to provide a general-purpose two-dimensional sensor in which the position and size of the measurement electrode can be arbitrarily set according to the sample and a measurement device using the two-dimensional sensor.

[Brief description of drawings]

FIG. 1 is a block diagram of a cell activity measuring apparatus according to an embodiment of the present invention.

FIG. 2 is a side sectional view (schematic diagram) and a plan view of a two-dimensional sensor used in the cell activity measuring apparatus of FIG.

FIG. 3 is a diagram showing an equivalent circuit of a circuit in which a photo-AC current flows in the measuring apparatus of FIG.

FIG. 4 is a waveform diagram showing an example of the measured optical AC current and changes in its amplitude.

FIG. 5 is a block diagram of a pH measurement circuit using LAPS related to the present invention.

FIG. 6 is a graph showing how the photo-AC current vs. bias voltage characteristic obtained from the measurement circuit of FIG. 5 shifts depending on the pH.

[Explanation of symbols]

 1 Two-dimensional sensor 1a Working electrode thin film 1b Enclosure 2 Sample (cell) 3 Incubator part 4 Temperature control unit 5 Heater / fan unit 6 Flowmeter 7 Solenoid valve 8 Potentiostat 9 Amplifier 10 Computer 11 Laser light source 12 Light source driving device 13 XY stage

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yu Iwasaki 8-14 Higashikari Shinmachi, Hirakata City, Osaka Prefecture

Claims (7)

[Claims] 1. A thin film for a working electrode is vapor-deposited on the Si-side back surface of a sensor substrate composed of three layers of Si, SiO ₂ , and Si ₃ N ₄ , and a sample cell, a culture, is deposited on the Si ₃ N _4- side surface. liquid,
And a reference electrode is provided with an enclosure, and in the planar distribution of the potential change generated by the activity of cells placed inside the enclosure, a signal mainly corresponding to the potential change of the portion irradiated with light is the working electrode. A two-dimensional sensor for measuring cell activity, which is characterized by being taken out from a cell. 2. The cell activity measuring device according to claim 1
A DC bias voltage is applied between the two-dimensional sensor, a laser light source for irradiating a laser beam on the back surface thereof, a working electrode on the back surface side of the two-dimensional sensor for measuring cell activity, and a reference electrode placed in an enclosure on the front surface side. A cell activity measuring device comprising a direct current power source to be applied and a processing means for processing a signal obtained between the both electrodes. 3. The cell activity measuring device according to claim 2, further comprising a culture maintaining means for maintaining a culture environment of the cells placed in the enclosure of the two-dimensional sensor for measuring cell activity. 4. A light source driving means for driving the laser light source at a high frequency to irradiate a high frequency modulated light is provided, and the processing means is configured to detect a change in amplitude of an optical alternating current flowing between the both electrodes. The cell activity measuring device according to claim 2. 5. The cell activity measuring device according to claim 2, further comprising means for scanning the laser beam emitted from the laser light source within a predetermined range on the back surface of the two-dimensional sensor for measuring cell activity. 6. The laser light source is provided with a laser array in which a plurality of lasers for irradiating the back surface of the two-dimensional cell activity measuring sensor with laser light are arranged in a two-dimensional matrix. The cell activity measuring device according to claim 2. 7. The cell activity measuring device according to claim 2, further comprising an XY stage for controlling the position of the two-dimensional sensor for measuring cell activity in the horizontal direction.